Size-Dependent Toxicity of Silver Nanoparticles to Bacteria,Yeast, Algae,Crustaceans and Mammalian Cells In Vitro

The concept of nanotechnologies is based on size-dependent properties of particles in the 1–100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC₅₀ values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20–80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO₃). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for “size-dependent“ biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Toxicology analysis by means of the JSM-method

MOTIVATION: A model for learning potential causes of toxicity from positive and negative examples and predicting toxicity for the dataset used in the Predictive Toxicology Challenge (PTC) is presented. The learning model assumes that the causes of toxicity can be given as substructures common to positive examples that are not substructures of negative examples. This assumption results in the choice of a learning model, called the JSM-method, and a language for representing chemical compounds, called the Fragmentary Code of Substructure Superposition (FCSS). By means of the latter, chemical compounds are represented as sets of substructures which are 'biologically meaningful' from the expert point of view. RESULTS: The chosen learning model and representation language show comparatively good performance for the PTC dataset: for three sex/species groups the predictions were ROC optimal, for one group the prediction was nearly optimal. The predictions tend to be conservative (few predictions and almost no errors), which can be explained by the specific features of the learning model. AVAILABILITY: by request to finn@viniti.ru; serge@viniti.ru, http://ki-www2.intellektik.informatik.tu-darmstadt.de/~jsm/QDA.     

Role of feeder cells in spreading and cytoskeleton organization of newborn rat keratinocytes.

We studied the effect of feeder cells (fibroblasts) and a mixture of the extracellular matrix components, Matrigel, on spreading and cytoskeleton organization of newborn rat keratinocytes (REK). REK formed lamellipodia on being plated together with feeder cells and on the Matrigel as a substrate whereas the same REK plated alone on a plastic surface formed filopodia. REK lamellipodia formation in co-cultures depended on the fibroblast addition time. Although conditioned medium from fibroblast cultures was not enough to induce lamellipodia, the extracellular matrix left after fibroblast removal was as effective as Matrigel. Our results indicate that lamellipodia formation seems to depend on the factor(s) secreted by fibroblasts and associated with the extracellular matrix.     

The effect of low temperatures on the viability of human epidermal keratinocytes found at different stages of differentiation

The aim of this study was a comparative analysis to the degree of stability of human epidermal cells found at different stages of differentiation to low temperatures. The effect of different subzero temperatures of liquid nitrogen vapor on keratinocytes found both in human skin fragments and as isolated cells extracted from skin fragments has been studied. The degree of stability of epidermal cells low temperatures was evaluated by their ability to form a multilayer stratum in culture; hence this phenomenon explains the survival of a sufficient amount of proliferative cells after exposure to subzero temperatures. Quantitative analysis of the ratio of epidermal stem, transitory and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined using antibodies corresponding to their different stages of differentiation. The results of this research show that the stability of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. Epidermal stem cells and transitory cells are more stable than differentiated cells.     

Effect of protein synthesis inhibitors on DNA replication. I. Effect of the short-term action of cycloheximide on cell permeability for thymidine in vitro]

The effect of protein synthesis inhibitors on DNA replication was studied on L cells. After a 10 minutes' action of the inhibitors, protein synthesis was seen to be completely blocked, and DNA synthesis decreased by 85%. Four hours after a 20-minutes' cycloheximide treatment, the cells completely restored their ability to protein synthesis and DNA replication and even surpass the control level, due, probably, to a partial cell synchronization in S period. The short action of cycloheximide did not interfere with thymidine uptake by the cells. The rate of the exogenous precursor uptake was even higher than that in the control, apparently, because of its much reduced utilization in the inhibited DNA synthesis.     

Dynamics of a 3H-lysine pulse label in the culturing of L cells

Using autoradiography, 3H-lysine, 3H-thymidine, and 3H-tryptophane puls labeled L-cells were examined as long as through four passages. Our studies demonstrate that no renewal of 3H-lysine labels takes place in chromosomes and nuclei. Unlike, the cytoplasmic labels of 3H-lysine and chromosomal, nuclear and cytoplasmic labels of 3H-tryptophane showed an intensive renewal. A question of renewal of lysine-rich histones is discussed.     

Autoradiography of lysed L cells with labelled 3H-lysine]

Using the method of fibr-DNA autoradiography, 3H-lysine labeled L cells were examined. Fibr-labeled structures as long as 400 mkm were revealed. The pattern of labeling along fibres was same as when cells were labeled with 3H-thymidine. In cross experiments when 3H-lysine and 3H-thymidine were used simultaneously, the pattern of labeling remained the same. The data obtained may evidence that the complexation of new synthesized histones with DNA takes place synchronously with the replication, and in the points of chromosome replication.